Inflammation is the body's innate response to tissue injury and infection. The inflammatory response is critical for fighting infections and healing wounds. However, in most diseases damage to normal tissues is due to inflammation that does not resolve or is directed against the host. Inflammatory stimuli activate endothelium of local vessels to express adhesion molecules that recruit circulating leukocytes from the blood. This occurs in a series of steps involving interactions between these adhesion molecules and counter-receptors on the leukocytes. Transendothelial Migration (TEM) is arguably the most critical step in the inflammatory response since it is essentially irreversible. PECAM and CD99 are two molecules important the execution of this process. Interfering with the function of these molecules blocks TEM both in vitro and in vivo. CD99L2 is a recently-described membrane protein that is related to CD99. CD99L2 is expressed at the borders of endothelial cells and on the surface of leukocytes. Blocking CD99L2 using antibodies significantly blocks recruitment of neutrophils and monocytes in mice. Thus, CD99L2 has been shown to be another important molecule regulating TEM; however, how it regulates this process is not known. PECAM regulates a step in TEM that is upstream of the step regulated by CD99. How CD99L2 functions in relation to PECAM and CD99 is not known. Furthermore, the studies on CD99L2 have all been done in mice. My preliminary data demonstrate for the first time a role for CD99L2 in TEM in human cells. My project will determine the role of CD99L2 in TEM of human leukocytes across human endothelial cells. CD99 and PECAM are expressed in an interconnected network of membrane vesicles just underneath the plasma membrane known as the lateral border recycling compartment (LBRC). Membrane from this compartment is trafficked to the sites of transmigrating leukocytes in a process known as targeted recycling (TR). This supplies the migrating leukocyte with adhesion molecules and additional membrane surface area to facilitate its movement across the endothelial cell. TR of the LBRC is essential for TEM; anything that blocks TR blocks TEM. Whether CD99L2 plays a role in this process is not known. My project will determine whether CD99L2 is in the LBRC, is involved in TR, and the mechanism by which it signals to carry out its role in TEM. It is also important to validate our i vitro findings in vivo. I will do this by directly determining the role of CD99L2 in real time in lving mice using spinning disc intravital microscopy. I will first observe leukocytes interacting with endothelial cells in the cremaster circulation of CD99L2 deficient mice to determine if the step in TEM that is regulated by CD99L2 is the same in vivo as we found in vitro. I will also determine whether the mechanism of action of CD99L2 in vivo is the same as we found in vitro. The successful conclusion of these studies will provide insight into the mechanisms of TEM and identify CD99L2 as a novel therapeutic target for a multitude of diseases caused by pathologic inflammation.